U.S. patent application number 08/796043 was filed with the patent office on 2002-05-23 for transduced human hematopoietic stem cells.
Invention is credited to HARDY, STEVE, JORDAN, CRAIG T..
Application Number | 20020061293 08/796043 |
Document ID | / |
Family ID | 27359380 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020061293 |
Kind Code |
A1 |
JORDAN, CRAIG T. ; et
al. |
May 23, 2002 |
TRANSDUCED HUMAN HEMATOPOIETIC STEM CELLS
Abstract
The present invention describes a novel subset of human
hematopoietic stem cells that are defined by the ability to express
transduced genetic material, and methods for producing the same.
The transduced hematopoietic cells are preferably comprised of
primary human CD34.sup.+ cells.
Inventors: |
JORDAN, CRAIG T.;
(LEXINGTON, KY) ; HARDY, STEVE; (SAN FRANCISCO,
CA) |
Correspondence
Address: |
ROYLANCE, ABRAMS, BERDO & GOODMAN, LLP
1300 19TH STREET, NW
SUITE 600
WASHINGTON
DC
20036-2680
US
|
Family ID: |
27359380 |
Appl. No.: |
08/796043 |
Filed: |
February 5, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60011172 |
Feb 5, 1996 |
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60011219 |
Feb 6, 1996 |
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60021683 |
Jul 12, 1996 |
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Current U.S.
Class: |
424/93.21 ;
435/320.1; 435/325; 435/455; 435/69.1; 514/44R |
Current CPC
Class: |
A61P 31/18 20180101;
A61P 37/00 20180101; C12N 15/86 20130101; C12N 5/0647 20130101;
C12N 2710/10343 20130101; A61P 31/00 20180101; A61K 38/00 20130101;
A61K 48/00 20130101 |
Class at
Publication: |
424/93.21 ;
514/44; 435/325; 435/455; 435/69.1; 435/320.1 |
International
Class: |
A61K 048/00 |
Claims
1. Transduced human CD34.sup.+ cells comprising recombinant genetic
material of interest, said cells having been transduced absent a
period of selective culture and having the property of being
capable of expressing the genetic material of interest.
2. Transduced cells according to claim 1 wherein said cells have
been transduced by a replication deficient virus comprising the
genetic material of interest.
3. Transduced cells according to claim 2 wherein said virus is an
adenovirus.
4. Transduced cells according to claim 3 wherein said cells express
the genetic material of interest in vitro.
5. Transduced cells according to claim 1 wherein said expressing is
in vivo.
6. The use of transduced human CD34.sup.+ cells according to claim
1 to effect a therapeutic benefit to a human patient.
7. The use according to claim 6 wherein said cells express a
recombinantly encoded cytokine.
8. The use according to claim 7 wherein said cytokine is Stem Cell
Factor.
9. The use according to claim 7 wherein said cytokine is
GM-CSF.
10. The use according to claim 7 wherein said cytokine is
G-CSF.
11. The use according to claim 7 wherein said cytokine is IL-3.
12. The use according to claim 7 wherein said cytokine is
erythropoietin.
13. The use according to claim 7 wherein said cytokine is IL-6.
14. The use according to claim 7 wherein said cytokine is Flt-3
ligand.
15. A method of making transduced human CD34.sup.+ hematopoeitic
stem cells, comprising: a) culturing said cells in the presence of
IL-3, IL-6, and stem cell factor; and b) infecting said cells with
a purified and concentrated replication deficient chimeric
adenovirus containing a gene encoding a pharmaceutically active
product.
Description
1.0. INTRODUCTION
[0001] The present invention relates to human cell biology, and
discloses a novel subset of CD34.sup.+ human hematopoietic stem
cells defined by their ability to express recombinant genetic
material which has been introduced to the cells by any of a variety
methods. The present application claims priority to U.S.
Provisional Applications Ser. Nos. 60/011,172, filed Feb. 5, 1996;
60/011,219, filed Feb. 6, 1996; and 60/021,683, filed Jul. 12,
1996.
2.0. BACKGROUND
[0002] Many human tissues such as skin, blood, and internal
epithelial tissue are composed of relatively short-lived cells.
Because these cells are short-lived, the body must constantly
regenerate these cells. Stem cells are a special class of cells in
the body that may either divide symmetrically to produce two
identical stem cells or asymmetrically to produce one new stem cell
and one determined or fully differentiated cell (which will replace
the short-lived cell). Accordingly, in order to maintain these
tissues, the body must ensure that a carefully regulated supply of
stem cells is maintained throughout life.
[0003] Additionally, stem cells may be totipotent (i.e., germ line
stem cells), pluripotent (i.e., CD34.sup.+ hematopoietic stem
cells), or unipotent (i.e., CD10.sup.+ lymphoid progenitor
cells).
[0004] During mammalian embryogenesis, hematopoietic stem cells
migrate via the bloodstream to the liver and spleen to seed these
tissues, which then carry the burden of hematopoiesis until birth
and for some time thereafter. Foci of hematopoiesis can been
detected in the liver by the sixth week, although in fetal rats the
spleen, along with the mesentery and intestine, appears to have a
specific affinity for inoculated adult lymphocytes from the
earliest gestational age examined (Chen and McCullagh (1992) J.
Reprod. Immunol. 22:127-141). Erythropoiesis predominates in the
fetal liver and spleen, although some granulopoiesis also occurs.
Hematopoiesis in the fetal liver and spleen does not involve
synchronous cell growth, and results enucleated blood cells.
Synthesized hemoglobin is not limited to the embryonic type.
[0005] The fetal spleen transiently serves as a hematopoietic organ
between the third and fifth months of gestation. Pre-B cells
(CD24.sup.+, sIgM.sup.-) can be detected in the human fetal spleen
by 12 weeks (Solvason and Kearney (1992) J. Exp. Med. 175:
397-404).
[0006] Several studies have begun to elucidate, at a molecular
level, some of the processes of cell migration and hematopoietic
maturation and differentiation occurring in the fetal spleen.
Recombinant human granulocyte colony stimulating factor (rhG-CSF)
administered to pregnant rats has been shown to cross the placenta
and specifically induce bone marrow and spleen myelopoiesis in the
fetus and neonate. The fetal and neonatal spleen displays an
exquisite degree of developmental sensitivity to this cytokine,
which results in increased white blood cell counts due to
circulating numbers of polymorphonuclear (PMN) cells and increasing
the number of post-mitotic (PMN, bands, and metamyelocytes) and
mitotic (promyeloblasts, myeloblasts, and metamyeloblasts) myeloid
cells in the spleens of neonates (Medlock et al. (1993) Blood
81:916-922). The majority of B cells in the fetal human spleen
express, inter alia, CD5 and CD10 and can be induced to produce
IgM, IgG, IgG4, and IgE (but not IgA) in response to IL-4 in the
presence of anti-CD40 monoclonal antibody (mAb) or cloned CD4 T
cells (Punnonen et al. (1992) J. Immunol. 148:398-404). It has also
been demonstrated that fetal splenic mononuclear cells produce IL-2
and IL-6. Levels of IL-2 and IL-6 increase with gestational age and
correlate positively with natural killer (NK) cell activities (Lu
et al. (1992) Shih Yen Sheng Wu Hsueh Pao (China) 25:305-309).
[0007] Eventually, the site of hematopoiesis is transferred to the
bone marrow, which is predominantly granulopoietic. Beginning at
the second month of fetal development, the bone marrow plays an
increasingly important role in hematopoiesis, becoming the
predominant site for hematopoiesis by the second half of gestation.
After birth, the bone marrow is eventually the only hematopoietic
organ, although both the liver and the spleen can serve as sites of
extramedullary hematopoiesis if the bone marrow fails. For a recent
review of embryonic and fetal hematopoiesis, see Tavassoli (1991)
Blood Cells 17:269-281. An interesting feature of developmental
hematopoiesis is that CD34.sup.+ stem cells that are introduced
into the bloodstream will eventually "home" to the bone marrow.
[0008] Because stem cells are essentially immortal within the body,
they constitute a particularly desirable target for human gene
therapy. Where the recombinantly encoded product is preferably
delivered to the bloodstream, hematopoietic stem cells (e.g.,
CD34.sup.+ cells) are particularly attractive targets for gene
delivery. Unfortunately, the relatively low number of CD34.sup.+
hematopoietic stem cells in the body, as well as the inherent
inefficiency of several prior transduction systems (i.e., calcium
phosphate transfection, etc.) had made the efficient targeting and
transfection of CD34.sup.+ hematopoietic stem cells somewhat
difficult. This has proved to be especially true where viral
vectors have been employed for gene delivery because of low
inherent infectabilty or the presence of toxic contaminants within
the viral preparations, and the inherent toxicity that is often
associated with the exposure of cells to high titer virus
stocks.
[0009] An additional consideration is that some viral vectors
require that the target cells are either actively replicating, or
at a given stage of the cell cycle, in order to efficiently express
the recombinant genetic material of interest. Moreover, the
presence of certain cellular receptors may also be required for the
efficient infection and delivery of genetic material of interest to
hematopoietic stem cells.
[0010] An additional consideration when targeting CD34.sup.+
hematopoietic stem cells for gene delivery is that the expression
of the introduced recombinant genetic material may prove somewhat
variable. The presently observed differential expression of
introduced genetic material within the CD34.sup.+ cell population
defines a new subgroup, or tropism, within the population which may
prove particularly important for gene delivery applications.
[0011] Adenovirus have proved to be of particular interest for the
viral transduction of stem cells because of several features of
adenoviral biology (See generally, Berkner, K. L. (1992) Curr. Top.
Microbiol. Immunol. 158:39-66). For instance, viral concentration,
or titer, is often an important factor in achieving high efficiency
transduction of mammalian cells. Adenovirus, by virtue of their
life-style, generally allow growth conditions which result in
production of higher titer stocks then other mammalian virus.
[0012] Also unlike other viruses, adenovirus capsids are not
enveloped. Because of this fact, adenovirus particles are quite
stable, and may retain infectivity after any of a variety of
laboratory procedures. Procedures of particular interest include
methods of concentrating infective virus, e.g., CsCl
centrifugation, or methods that allow virus to be stored for
relatively long periods while retaining substantial
infectivity.
[0013] Furthermore, the expression of genes encoded by recombinant
adenovirus does not require target cell proliferation or viral
integration, although a small subset of the adenovirus presumably
integrate into the host genome during infection. Hence, adenoviral
vectors are generally better suited than other viral vectors for
the transduction of postmitotic, slowly proliferating, or
nonreplicating cells.
[0014] Additionally, particularly where species-specific infection
is preferred, replication deficient human, or murine, adenovirus
are available for the construction of recombinant virus particles
that express a gene of interest. Thus, unlike transduction systems
using other eucaryotic virus vectors, recombinant adenovirus can be
engineered to utilize viral coat proteins which normally facilitate
the normal infection of human cells or cells of other species,
rather then rely on the viral coats of a less specific, or
amphotropic, nature. This species specificity appears to result in
more efficient infection kinetics than can generally be obtained by
virus with less specific infectivity.
[0015] An additional advantage of using adenovirus for gene
delivery is that the genetic material transduced (to be expressed)
into the host cell is DNA. Thus, expression of the transduced gene
does not need to be preceded by reverse transcription. This is
particularly advantageous where the intended recipient is
undergoing treatment for the suppression of retroviral disease
(i.e., AZT treatment to inhibit reverse transcriptase activity),
such as treatment for acquired immunodeficiency syndrome
(AIDS).
[0016] Recombinant adenoviral vectors have been generated which
express a variety of genes. Perhaps most notable is the replication
deficient adenovirus vector Ad.RSV that expresses incorporated
genetic material of interest using an incorporated promoter from
the Rous Sarcoma Virus. In particular, Ad.RSV beta gal (which
expresses the bacterial .beta.-galactosidase gene) has been used as
a marker for in vivo gene transfer experiments involving salivary
glands (Mastrangeli et al. (1994) Am. J. Physiol. 266:1146-1155);
mesothelial cells (Setoguchi et al. (1994) Am. J. Respir. Cell.
Mol. Biol. 10(4):369-377); and tumor cells (Brody et al. (1994)
Hum. Gene Ther. 5(4):437-447, Chen et al. (1994) Proc. Natl. Acad.
Sci., U.S.A. 91(8):3054-3057).
[0017] In general, adenoviral transduction results in a more
transient expression of the inserted genetic material relative to
other viral gene delivery systems (i.e., retrovirus, and
adeno-associated virus).
3.0. SUMMARY OF THE INVENTION
[0018] The subject invention provides for methods and processes for
the identification, isolation, and use of a novel subclass of
CD34.sup.+ human hematopoietic stem cells that are capable of being
transfected with a chimeric adenovirus to express recombinant
genetic material of interest.
[0019] Accordingly, an important embodiment of the present
invention is a CD34.sup.+ stem cell that has been virally
transduced with a recombinant virus, preferably adenovirus, such
that the transduced cells is capable of expressing the transduced
genetic material of interest in vivo.
[0020] Preferably, the titer and infectivity of the transducing
virus will be sufficient to allow efficient transduction of the
claimed human CD34.sup.+ cells without requiring a period of
selective culture to preferentially expand the number of transduced
cells. Accordingly, the transducing virus used in the present
invention need not comprise or encode a functional selectable
marker.
[0021] After being transfected with a vector encoding a suitable
gene product, or tagging with an appropriately labeled antibody or
receptor specific for the subclass, the novel high-expressing
subclass of transduced CD34.sup.+ hematopoietic stem cells may be
sorted and isolated for further manipulation or study using any of
a variety of well known techniques including fluorescence activated
cell sorting (FACS), centrifugation, or the like.
4.0. DESCRIPTION OF THE FIGURES
[0022] FIG. 1 is a titration curve of alkaline phosphatase
expression in the target CD34.sup.+ cell population as a function
of the multiplicity of infection of input recombinant
adenovirus.
[0023] FIG. 2 shows the quasi transient nature of recombinant gene
expression after the target CD34.sup.+ cell population was
enzymatically tagged with a recombinant adenovirus comprising the
lacZ gene. The graph shows the diminution of lacZ activity over 8
days post infection.
[0024] FIG. 3 compares the levels of alkaline phosphatase
expression in populations of: uninfected CD34.sup.+ cells (FIG.
3A), or CD34.sup.+ cells that have been infected with a recombinant
adenovirus encoding an alkaline phosphatase gene, (FIG. 3B).
[0025] FIG. 4 compares the levels of alkaline phosphatase
expression in populations of quiescent (G.sub.0) CD34.sup.+ cells
using either uninfected CD34.sup.+ cells (FIG. 4A), or CD34.sup.+
cells infected with a recombinant adenovirus encoding an alkaline
phosphatase gene expressed (FIG. 4B).
5.0. DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention allows for the efficient and practical
identification and isolation of primary human CD34.sup.+ stem cells
that are permissive for the uptake and expression of genetic
material of interest. As used herein, the term "expression" refers
to the transcription of the DNA of interest, and the splicing (if
any), processing, stability, and, optionally, translation of the
corresponding mRNA transcript.
[0027] The genetic material of interest can optionally comprise a
gene, or fraction thereof, oriented to express either a polypeptide
or protein of interest, a "sense" or "antisense" nucleic acid of
structural or regulatory importance, or a functional ribozyme.
Preferably, such nucleic acid products will be pharmacuetically
active or shall provide a therapeutic benefit to the patient.
[0028] Preferably, the DNA of interest will be placed in an
expression cassette that contains a eucaryotic promoter and/or
enhancer region, an insertion site for the genetic material of
interest, and a substantially noncoding 3' DNA which facilitates
the stability, polyadenlyation, or splicing of the transcript. Any
number of transcriptional promoters and enhancers may be used in
the expression cassette, including, but not limited to, the herpes
simplex thymidine kinase promoter, cytomegalovirus (CMV)
promoter/enhancer, SV40 promoters, and retroviral long terminal
repeat (LTR) promoter/enhancers. Of special interest are any of a
number of well characterized retroviral promoters, particularly the
Moloney murine leukemia virus (MLV) LTR promoter and the human
immunodeficiency virus (HIV) LTR.
[0029] The genetic material of interest will generally constitute
nucleic acid which encodes pharmaceutically active products or
proteinaceous material. Preferably, the pharmaceutically active
protein will provide a therapeutic benefit to the patient. The
expressed form of the encoded proteinaceous material may or may not
comprise sugar residues. The encoded genetic material of interest
may encode a product which is useful in both human or veterinary
medicine, either by way of treatment or prophylaxis of diseases or
their symptoms, or is useful cosmetically or diagnostically.
[0030] Although virtually any DNA sequence of interest may be
expressed in CD34.sup.+ stem cells, particular examples of encoded
genetic material of interest which may be used in accordance with
this invention include, but are not limited to, protein hormones
such as insulin, calcitonin and growth hormone, erythropoietin,
plasminogen activators and their precursors, such as t-PA,
urokinase, G-CSF, GM-CSF, stem cell factor (SCF) or other
cytokines, pro-urokinase and streptokinase, interferons including
human interferon alpha, interleukins including IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, and blood factors including Factor
VIII. Additionally, the claimed CD34.sup.+ cells may be used to
express virtually any proteinaceous endocrine hormones as well as
any proteinaceous cell or viral receptors.
[0031] Given their crucial role in hematopoiesis, the claimed
CD34.sup.+ stem cells may also prove useful as targets for the
delivery of genes encoding anti-malarial factors, gene therapy
(i.e. adenosine deaminase deficiency, or .beta.-thalassemia, etc.),
or, where vectors targeting specific gene replacement are used, as
a means of correcting gene defects that are associated with sickle
cell anemia.
[0032] Optionally, suicide sequences may also be stably and
quiescently inserted into stem cells such that the cells will only
express the toxic suicide gene upon activation by a specific
stimuli. For example, the gene encoding the polio virus translation
inhibition protein may placed under the transcriptional control of
the HIV LTR promoter and inserted into the target stem cell. The
HIV promoter will remain inactive until the cells are infected with
HIV virus which expresses the appropriate trans-acting
transcription factors that induce the HIV LTR mediated expression
of the suicide product. After the suicide product is expressed, the
cells die and the further spread of the virus is effectively
eliminated.
[0033] Many cytokines and factors are toxic when used systemically.
By using transduced stem cells as bioreactors to produce and
deliver a protein or peptide of interest to the body, one can
effectively deliver local concentrations of factor (in the vicinity
of the transduced cells) while effectively maintaining very low
systemic concentrations of factor (thus avoiding much of the
systemic toxicity effects. This feature of the present invention is
particularly important given the fact that infused CD34.sup.+ stem
cells will "home" to the bone marrow. Accordingly, the claimed
transduced stem cells may be used to produce and target various
therapeutic agents to the bone marrow. Thus, a particularly useful
embodiment of the presently disclosed invention is the used of the
claimed cells to produce and deliver erythropoietin (or other
cytokines) to the bone marrow. Depending on the mode of
transduction used, factor production and delivery may be permanent
or temporary.
[0034] An additional feature of the present mode of factor
production and delivery is that one is able to avoid the lengthy
purification, formulation, and packaging processes that are
typically required where direct introduction of pharmaceutical
compositions comprising purified factors is contemplated.
[0035] Although enduring, or permanent, expression of the genetic
material of interest may often be preferred, there are many
instances where transient expression of recombinant genetic
material of interest is more desirable. For example, transient
expression may be preferred where one is simply delivering a viral
receptor to the stem cells in order the increase or enhance the
infectivity of transducing virus that will integrate and stably
express a cloned genetic material of interest (e.g., retrovirus or
adeno-associated virus).
[0036] Additionally, transient transfection and expression may be
particularly preferable where acute diseases are involved. For
example, by inserting the proper drug resistance factor, transduced
CD34.sup.+ stem cells may be temporarily rendered immune to
specific antibiotic or chemotherapeutic agents. Given that the
hematopoietic system, is often adversely impacted by the effects of
chemotherapeutic treatment, CD34.sup.+ stem cells may be transduced
to transiently express factors that enhance the cells', and
surrounding cells', resistance to a given chemotherapeutic
agent.
[0037] Aplastic anemia is a grave complication that may accompany
treatment with a variety of therapeutic agents (i.e.,
chloramphenicol, inter alia). Thus, the introduction of suitable
drug resistance genes into CD34.sup.+ cells, in vivo or in vitro,
may protect these cells from the potentially harmful side-effects
of otherwise therapeutic agents.
[0038] Similarly, a regulatory gene or antisense nucleic acid may
be delivered into the CD34.sup.+ cell population which transiently
and reversibly disrupts stem cell division or DNA synthesis during
the period in which the patient is exposed to high doses of
chemotherapy or ionizing radiation.
[0039] The terms substantially arresting or substantially
inhibiting DNA synthesis shall generally mean that the net
level/amount of DNA synthesis in treated cells be at least about 70
percent that of control or untreated cells, and preferably mean
that the level of DNA synthesis shall be about 50 percent that of
control cells. optionally, the extent of DNA synthesis may be
calculated on a per viable cell basis, and normalized
accordingly.
[0040] Similarly, the use of transient transduction to reversibly
inhibit DNA synthesis may effectively render the growth of the
target cell population substantially synchronous. For the purposes
of this application, the term substantially synchronous population
of cells shall mean that generally at least about 50 percent more
of the cells in a given cell population will be at or in the same
stage of cell division at a given point of interest; preferably at
least about 75 percent more of the cells in a given population are
at or in the same stage of cell division; and optimally at least
about 100 percent more of the cells in a given population will be
at or in the same stage of cell division as compared to untreated
control cells.
[0041] One of ordinary skill will appreciate that, from a medical
practitioner's or patient's perspective, virtually any alleviation
or prevention of an undesirable symptom (e.g., symptoms related to
disease, sensitivity to environmental or factors, normal aging, and
the like) would be desirable. Thus, for the purposes of this
Application, the terms "treatment", "therapeutic use", or
"medicinal use" used herein shall refer to any and all uses of the
claimed cells which remedy a disease state or symptoms, or
otherwise prevent, hinder, retard, or reverse the progression of
disease or other undesirable symptoms in any way whatsoever.
[0042] When used in the therapeutic treatment of disease, an
appropriate dosage of transducing virus (when targeting CD34.sup.+
cells in vivo) or in vitro transduced stem cells or derivatives
thereof, may be determined by any of several well established
methodologies. Where toxicity is a concern, animal studies are
commonly used to determine the maximal tolerable dose, or MTD, of
bioactive agent per kilogram weight. In general, at least one of
the animal species tested is mammalian. Those skilled in the art
regularly extrapolate doses for efficacy and avoiding toxicity to
other species, including human. Before human studies of efficacy
are undertaken, Phase I clinical studies in normal subjects will
help establish safe doses. Preferably, the transducing virus will
be prepared such that it is substantially non-toxic to the target
cells at high multiplicities of infection (m.o.i., generally
exceeding about 100, often exceeding 250, and preferably exceeding
about 500 viral particles per CD34.sup.+ target cell with at least
about 50 percent, and preferably at least about 80 percent of the
target cells (and/or supporting stromal cells) remaining viable
after exposure to the transducing virus).
[0043] Where diagnostic, therapeutic or medicinal use of the
transduced stem cells, or derivatives thereof, is contemplated,
that transduced stem cells to be reimplanted in vivo will generally
be tested for sterility (absence of mycoplasma, bacteria, fungus,
or other potential pathogens), viability, expression of the gene of
interest, and for the presence of recombinant viral sequence, and
absence of replication competent helper virus. Gene modified stem
cells may be introduced in vivo by any of a number of established
methods, but preferably by intravenous (I.V.) infusion.
[0044] Given that the presence of contaminating helper virus
genomes may result in the production of replication competent viral
particles in vivo, the chimeric viral particles used to infect the
cells intended to provide a therapeutic benefit in vivo will be
substantially helper free. For the purposes of this application,
the term substantially helper free shall mean that the supernatants
from target cells infected with 1 ml undiluted preparation of a
given chimeric virus preparation shall not contain significant
levels of replication competent virus as identified by plaque
assays (i.e., typically less then about ten percent of the
infectious plaque activity of comparative titers of replication
competent virus). Alternatively, the viral vectors and/or
helper-virus may be engineered to incorporate a latent suicide gene
(e.g., herpes simplex virus thymidine kinase) that may be activated
to kill cells harboring the recombinant virus).
[0045] Although the use of chimeric adenovirus similar to those
described in U.S. application Ser. No. 08/311,485, filed Sep. 23,
1994, herein incorporated by reference, are used in the examples,
it is contemplated that additional vectors/methods that may be used
to deliver nucleotide sequences to the patient including, but are
not limited to, liposomal or lipid-associated delivery, direct
injection of nucleotides encoding the desired products, and the
like.
[0046] Additionally, other eucaryotic viruses that may be prove
useful in producing the claimed human CD34.sup.+ transduced stem
cells include papilloma virus, herpes virus, adeno-associated
virus, retrovirus, rabies virus, and the like (See generally,
Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., Vol. 3:16.1-16.89 (1989).
[0047] Optionally, human primary hematopoietic cells will be
transduced soon after isolation, and the claimed methods will not
require that the primary target cells be adapted to growth in
myeloid long-term culture (LTC), nor generally require long periods
of culture in vitro. Additionally, the claimed methods may not
require the use nor the support of fibroblast stromal or "feeder
cells" during the transduction procedure, nor to effect or assess
expression of the transduced genetic material.
[0048] The methods used to culture the stem cells prior to
transduction may dramatically effect transduction efficiency. In
particular, the presence of cytokines, or combinations thereof, is
deemed to be important in preconditioning the stem cells to allow
efficient transduction and expression. The cytokines or factors
employed during stem cell culture may be of natural, recombinant,
or synthetic origin. Although factors derived from other species
may be used, factors of human origin are preferred. Examples of
such cytokines or factors include, but are not limited to, LIF,
interleukins (including IL-1 through IL-14, and analogues thereof),
steel factor, colony stimulating factors (CSF), GM-CSF, G-CSF,
MIP-1.alpha., Flt-3 ligand, and the like.
[0049] Typically, such cytokines and growth factors will be used at
concentrations that are amenable to enhancing transduction while
not unduly toxic to the CD34.sup.+ stem cells. Significantly, the
presently described cytokines have proven capable of enhancing
adenoviral transduction when used at concentrations well below
those typically required to stimulate cell growth. Although the
amount of cytokine to be added to the culture will generally be
cytokine-specific, typically cytokine will be added to the stem
cell culture medium an a concentration of between about 1 ng/ml and
about 100 ng/ml, generally between about 5 ng/ml and about 50
ng/ml, and preferably between about 5 and about 30 ng/ml.
[0050] Generally, where IL-3 is added to the culture to enhance
adenoviral transduction it is present at a concentration ranging
between about 0.5 ng/ml and about 20 ng/ml, and preferably between
about 1 ng/ml and about 5 ng/ml. Generally, where IL-6 is used
during stem cell culture it present at a concentration ranging
between about 1 ng/ml and about 40 ng/ml, and preferably between
about 2 ng/ml and about 10 ng/ml.
[0051] Similarly, where SCF is added the culture it is used a
concentration ranging between about 2 ng/ml and about 100 ng/ml,
and preferably between about 5 ng/ml and about 25 ng/ml.
[0052] Where all three of IL-3, IL-6, and SCF are used to provide a
synergistic enhancement of adenoviral transduction of CD34.sup.+
stem cells, the cytokines are typically used at a respective ratio
of about 1:2:5, or any of the ranges consistent with the preferred
dose ranges for each cytokine as provided above.
[0053] Where the virally transduced cells are to be used in vivo,
the transducing recombinant virus, in addition to being
substantially helper free, may optionally be substantially
nonimmunogenic (i.e., does not express viral or other immunogenic
antigens to such an extent that a substantial immune response
against the transduced cells shall be generated in vivo.
[0054] Regardless of the specific means used to deliver the
recombinant nucleic acid of interest to the claimed transduced
CD34.sup.+ cells, the method of gene delivery will preferably be
sufficiently efficient that a substantial percentage of transduced
CD34.sup.+ cells may be obtained under nonselective conditions. For
the purposes of the present disclosure, the term "a substantial
percentage of transduced cells" shall generally mean that at least
about five percent of the net amount of CD34.sup.+ cells initially
exposed to the recombinant genetic material will be transduced to
express the genes of interest, preferably at least about twenty
percent of the CD34.sup.+ cells exposed to the recombinant genetic
material will be transduced to express the genes of interest,
specifically at least about thirty percent of the CD34.sup.+ cells
exposed to the recombinant genetic material will be transduced to
express the genes of interest. Given the relatively high efficiency
envisioned, the vectors used to deliver and guide the expression of
the genetic material of interest may contain a selectable marker,
but a selectable marker should not required to effect transduction
(i.e., the vectors may optionally lack a selectable marker).
[0055] The examples below are provided to illustrate the subject
invention. These examples are provided by way of illustration and
are not included for the purpose of limiting the invention in any
way whatsoever.
6.0. EXAMPLES
6.1. The Transduction of CD34.sup.+ Stem Cells
[0056] A recombinant adenoviral construct (AD-AP) that expresses
the alkaline phosphatase gene under the transcriptional control of
the Moloney murine leukemia virus (MLV) long terminal repeat (LTR)
was used to generate a stock of high-titer virus by standard
techniques (Graham and Prevec, 1991, Methods Mol. Biol.,
7:109-128). Typically, virus stocks were purified and concentrated
by, inter alia, CsCl centrifugation (followed by dialysis). The
above methods enable the production of adenoviral stocks of low
inherent toxicity with titers of at least about
1.times.10.sup.10/ml up to about 10.sup.13/ml.
[0057] Primary human CD34.sup.+ cells were isolated from human bone
marrow, mobilized peripheral blood, or umbilical cord blood using
standard procedures (Cellpro Ceprate Kit, or Miltenyi Biotech MACS
column). CD34.sup.+ cells were cultured in X-vivo 10 medium
(Biowhitaker) in the presence of one percent human serum albumin,
IL-3 (5 ng/ml), IL-6 (10 mg/ml), SCF (25 ng/ml), and five percent
fetal bovine serum.
[0058] The isolated cells were then infected with the isolated
virus at varying multiplicities of infection. The data obtained
from this experiment is shown in FIG. 1. FIG. 1 clearly indicates
that the percentage of virally transduced CD34.sup.+ cells (as
measured by detectable alkaline phosphatase expression) increases
as increasing amounts of virus are added to the cells.
Interestingly, the percentage of transduced cells seems to plateau
at about 40 percent.
[0059] This observation was repeatable and indicates that only a
discrete subset of the CD34.sup.+ cell population is able to be
transduced to express cloned genetic material introduced by the
chimeric adenoviral vectors used in the study.
[0060] The fact that only a fixed subset of CD34.sup.+ cells were
able to be transduced by adenoviral vectors may be explained by the
fact that only a fixed percentage of the stem cells were properly
conditioned in culture to be rendered transducible by adenovirus
vectors. For example, in order to achieve maximal levels of
transfection efficiency, the presently described human CD34.sup.+
cells must generally be exposed to human cytokines. In particular,
the presence of IL-3, IL-6, and stem cell factor (SCF), rapidly
increases transfection efficiency, and may even be deemed as
essential for the transfection of CD34.sup.+ stem cells. Each of
these cytokines enhance adenovirus transfection when used
individually, and, when combined, provide a synergistic effect that
typically enhances transfection efficiency by about 25 to about 50
percent greater beyond the mere additive effect of the individual
cytokines.
[0061] The discovery that cytokine exposure enhances the
transfectability of human CD34.sup.+ stem cells has far reaching
application. For example, it is likely that exposure to other
cytokines, and particularly mixtures thereof, will render higher
percentages of a CD34.sup.+ population transducible by adenoviral
vectors. Additionally, by using the proper cytokines, or
synergistic mixtures thereof, to similarly condition a population
of CD34.sup.+ cells, it is likely that the cells will also be
rendered more transducible by other means such as retroviral
vectors, adenoassociated virus vectors, lipofection,
electroporation, etc.
6.2. Transient Transduction of CD34.sup.+ Stem Cells With
AD-Lacz
[0062] A different adenoviral construct (AD-Lacz) that expresses
the lacZ gene was constructed and used in a time course experiment
that tracked the LacZ expression by the transduced CD34.sup.+ cells
over the course of eight days post-infection. The data resulting
from this experiment is shown in FIG. 2 which shows that the
percentage of transduced cells that expressed LacZ gradually
decreased after infection. Interestingly, the decrease was
nonlinear and may indicate that although the majority of the target
cells are presumably transiently infected, a portion of the target
cells may continue to express the introduced genetic material of
interest for substantial periods. The nonlinearity of the decrease
in expression may also be a function of the gradual dilution of the
replication deficient adenoviral encoded sequences as the
population of CD34.sup.+ cells gradually increased in the
culture.
6.3. Human CD34.sup.+ Stem Cells May be Transfected With AD-AP to
Express AP
[0063] FIG. 3A shows the results of a FACS analysis that was used
to directly quantify the extent to which uninfected CD34.sup.+
cells express alkaline phosphatase (AP). In this experiment, the
target cells were tagged with mouse anti-AP antibodies, and a
fluorescently labeled goat anti-mouse antibody. The data clearly
indicate that very little AP expression could be detected in
uninfected cells.
[0064] Conversely, the FACS analysis shown in FIG. 3B clearly
indicates that, after infection with AD-AP, a significant
percentage of the CD34.sup.+ cell population expresses the AP
gene.
[0065] 6.4. Quiescent Human CD34.sup.+ Stem Cells May be
Transfected With AD-AP to Express AP
[0066] FIG. 4A shows the results of a FACS analysis that was used
to directly quantify the extent to which uninfected quiescent
CD34.sup.+ cells express alkaline phosphatase (AP). In this
experiment, the target cells were tagged with mouse anti-AP
antibodies, and a fluorescently labeled goat anti-mouse antibody.
The data clearly indicate that very little AP expression could be
detected in uninfected cells.
[0067] Conversely, the FACS run shown in FIG. 4B clearly indicates
that, after infection with AD-AP, a significant percentage of the
quiescent CD34.sup.+ cell population expresses the AP gene.
[0068] Quiescent CD34.sup.+ cells were collected based on two
separate criteria. First, cells which did not express the Ki-67
antigen, a well known marker of cell activation (Schuler et al.,
1993, J. Cell Biol. 123(3):513-522). In addition, cells were
further selected which showed no evidence of DNA replication when
tested by staining with 7-amino-actinomycin-D (Rabinovitch et al.,
1986, J. Immunol. 136:2769).
[0069] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the above-described modes for carrying out
the invention which are obvious to those skilled in the field of
molecular biology or related fields are intended to be within the
scope of the following claims.
* * * * *